Immunoglobulin 2

ABSTRACT

The present invention relates to a method for the generation of single chain immunoglobulins in a mammal. In particular, the present invention relates to a method for the generation of single chain camelid VHH antibodies in a mammal which undergo the process of class-switching and affinity maturation found within antibody producing B cells. Single chain antibodies generated using the method of the present invention and the uses thereof are also described.

The present invention relates to a method for the generation of singlechain immunoglobulins in a mammal. In particular, the present inventionrelates to a method for the generation of single chain camelid VHHantibodies in a mammal. Single chain antibodies generated using themethod of the present invention and the uses thereof are also described.

BACKGROUND TO THE INVENTION

The antigen binding domain of an antibody comprises two separateregions: a heavy chain variable domain (VH) and a light chain variabledomain (V_(L): which can be either V_(kappa) or V_(lambda)). The antigenbinding site itself is formed by six polypeptide loops: three from VHdomain (H1, H2 and H3) and three from V_(L) domain (L1, L2 and L3). Adiverse primary repertoire of V genes that encode the VH and V_(L)domains is produced by the combinatorial rearrangement of gene segments.The VH gene is produced by the recombination of three gene segments, VH,D and J_(H). In humans, there are approximately 51 functional VHsegments (Cook and Tomlinson (1995) Immunol Today, 16: 237), 25functional D segments (Corbett et al. (1997) J. Mol. Biol., 268: 69) and6 functional J_(H) segments (Ravetch et al. (1981) Cell, 27: 583),depending on the haplotype. The VH segment encodes the region of thepolypeptide chain which forms the first and second antigen binding loopsof the VH domain (H1 and H2), whilst the VH, D and J_(H) segmentscombine to form the third antigen binding loop of the VH domain (H3).The V_(L) gene is produced by the recombination of only two genesegments, V_(L) and J_(L). In humans, there are approximately 40functional V_(k) segments (Schäble and Zachau (1993) Biol. Chem.Hoppe-Seyler, 374: 1001), 31 functional Vλ segments (Williams et al.(1996) J. Mol. Biol., 264: 220; Kawasaki et al. (1997) Genome Res., 7:250), 5 functional J_(κ) segments (Hieter et al. (1982) J. Biol. Chem.,257: 1516) and 4 functional J_(λ) segments (Vasicek and Leder (1990) J.Exp. Med., 172: 609), depending on the haplotype. The V_(L) segmentencodes the region of the polypeptide chain which forms the first andsecond antigen binding loops of the V_(L) domain (L1 and L2), whilst theV_(L) and J_(L) segments combine to form the third antigen binding loopof the V_(L) domain (L3). Antibodies selected from this primaryrepertoire are believed to be sufficiently diverse to bind almost allantigens with at least moderate affinity. High affinity antibodies areproduced by “affinity maturation” of the rearranged genes, in whichpoint mutations are generated and selected by the immune system on thebasis of improved binding.

The heavy chain locus contains a large number of variable chain genes(VH; in fact not complete genes but comprising a first coding exon plustranscriptional start site) that are recombined onto two short codingregions D and J (known as VDJ recombination) which procede the exonsthat code for the constant region of the heavy chain Cμ to give acomplete antibody heavy chain gene known as IgM. Subsequently a classswitch takes place where the variable part is recombined with anotherconstant region that is located downstream of the IgM constant region togive IgD, IgG, IgA and IgE (coded for by the exons of the various Cδ,Cγ, Cα, Cε located downstream of the gene for Cμ. The interveningconstant regions are deleted in the process. A similar process takesplace in the light gene loci, first the κ locus, and when this does notlead to a productive antibody in the λ locus (for review see Rajewski,K., Nature 381, p751-758, 1996; for an extensive review, see thetextbook Immunobiology, Janeway, C., Travers, P., Walport, M., Capra.J., Current Biology Publications/Churchill Livingstone/GarlandPublishing, fourth edition, 1999, ISBN 0-8153-3217-3).

Camelids (camels, dromedary and llamas) contain, in addition to normalheavy and light chain antibodies (2 light chains and 2 heavy chains inone antibody), single chain antibodies (containing only heavy chains).These are coded for by a distinct set of VH segments referred to as VHHgenes. Antigen binding for single chain antibodies is different fromthat seen with conventional antibodies, but high affinity is achievedthe same way, i.e. through hypermutation of the variable region andselection of the cells expressing such high affinity antibodies(affinity maturation). The VH and VHH are interspersed in the genome(i.e. they appear mixed in between each other). The identification of anidentical D segment in a VH and VHH cDNA suggests the common use of theD segment for VH and VHH. Natural VHH containing antibodies are missingthe entire C_(H)1 domain of the constant region of the heavy chain. Theexon coding for the C_(H)1 domain is present in the genome but isspliced out due to the loss of a functional splice acceptor sequence atthe 5′ side of the C_(H)1 exon. As a result the VDJ region is splicedonto the C_(H)2 exon. When a VHH is recombined onto such constantregions (C_(H)2, C_(H)3) an antibody is produced that acts as a singlechain antibody (i.e. an antibody of two heavy chains without a lightchain interaction). Binding of an antigen is different from that seenwith a conventional antibody, but high affinity is achieved the sameway, i.e. through hypermutation of the variable region and selection ofthe cells expressing such high affinity antibodies.

The structure of isolated VH domains has been determined using NMR andX-ray crystallography techniques (Spinell et al, (1996), Nat Structuralbiol. 3, 752). Data show that the Immunoglobulin fold is well preservedin Camelid VHH domains. Two beta sheets (one with four and one with fivebeta-strands) are packed against each other and stabilised by aconserved intradomain disulphide bond between C22 and C92. The side ofthe camel VHH domain corresponding to the VL interface of the normal VHin an Fv has a quite different architecture. Compared to the human VH,four amino acid substitutions are located in this region.

From a survey of all human and mouse VH antigen binding loop structures,it is apparent that there are only a restricted number of possibleconformations. Three and four different conformations are described forthe first and second antigen binding loop respectively. These canonicalstructures are determined by the length of the loop and the presence ofparticular residues at key positions. The H3 loop is extremely variablein length and sequence (Wu et al (1993) Proteins: structure, funct andgenet., 16, 1). Surprisingly, the antigen binding loop of camel VHdomains deviate from the canonical loop definitions of human and mouseVHH domains. This deviation could not be predicted as the loop lengthand the residues at the key positions are very similar between camel VHand human VH. The additional canonical loop structures in camel VHdomains make the structural repertoire of their paratope larger thanthat of VH domains in Fv fragments from conventional antibodies.Moreover, the hypervariable region around the first antigen binding loopis enlarged compared with human or mouse antibodies. It is thought thatthe extension of the first hypervariable region and concomitant enlargedantigen binding surface compared to that of a VH in a conventionalantibody compensates in part for the absence of a V_(L) domain(Riechmann, L. & Muyldermans, S (1999), 231 25-38).

A single domain camelid VHH antibody as well as being more suitable forstructural analysis than the larger heavy and light chain antibodymolecules, also provides a small and efficient antigen binding unit.Such an antibody has many and varied therapeutic potential. In addition,it has been found that camelid single chain antibodies can bind antigenswhich are inaccessible to antibodies possessing both heavy and lightchains. It is thought that this ability is due to the presence of alarge protruding third hypervariable loop of 10 amino acids or morewhich can insert into cavities of antigen surfaces. This is especiallysignificant as the catalytic site of an enzyme is often located at thelargest cavity on their protein surface. (Ladowski, R. A (1996). ProteinScience 5, 2438). Such sites are not normally immunogenic forconventional antibodies (Novotny, J et al, (1986) Proc Nat Acad Sci USA,83, 226). In the structure of the camel VHH cAb-Lys3, the 24 residue H3loop penetrates deeply into the active site of lysozyme (Transue, T. Ret al (1998) Prot: Structure, Funct and Genet, 32, 515), showing thatCamel heavy chain antibodies have the potential to form specific enzymeinhibitors.

Recently, isolated Camelid VHH domains have been generated in bacteria(Riechmann, L et al. Journal of Immunological Methods 231 (1999),25-38). However bacterial expression systems have the disadvantage thatthey do not perform post-translational modifications. Suchmodifications, in particular glycosylation events, are crucial for theeffective functioning of antibodies, particularly in an in vivoenvironment.

In the same study, the genes for Camel VHH domains were inserted intoexpression vectors and expressed in Cos cells to generate multi-domainproteins. In one example, an intact single heavy chain only antibody wasgenerated by cloning a particular camel VHH in front of the hinge andeffector function domains of human IgG1. The expression in Cos cells hasthe advantage over bacterial expression systems that post-translationalmodification events occur in these cells. Consistent with this was thefinding that these antibodies were fully active in antigen binding. TheDNA for the generation of these constructs is generally isolated frommature (i.e. those which have undergone affinity maturation) antibodiesgenerated from B cells. Although these single chain antibodies expressedin mammalian cells in an in vitro environment can bind to one or moreantigens, they cannot undergo the processes of class (isotype) switchingand affinity maturation (hypermutation). Thus the single chainantibodies expressed in Cos cells do not undergo the process of antibodyevolution as those naturally occurring antibodies generated within amammal. It is this process of antibody evolution which results in theproduction of specific antibodies which bind with high affinity. Thus,there remains a need in the art for a method allowing the generation ofsingle chain VHH antibodies in a mammal such that the normal processesof antibody evolution can take place.

In addition Camelid single chain antibodies have also been selected andexpressed using phage display technology. (Riechmann, L. & Davies, S. J.Biomol. NMR, 6, 141). Again though, the antibody constructs aregenerated from nucleic acid isolated from mature B cells or spleen, andtherefore as with the case above, the antibodies expressed do notundergo class switching and somatic hypermutation (affinity maturation)which is necessary for the production of specific antibodies which bindto their antigen with selectivity and high affinity.

The present inventors realised that if they could understand themechanism by which camelid single chain antibody molecules evolve (byclass-switching and affinity maturation) during early antibodydevelopment in B cells, then this system may be recreated in vivo. Thiswould allow the generation of vast quantities of an evolved single chainantibody for structural, therapeutic and diagnostic applications.

SUMMARY OF THE INVENTION

Antibody molecules which comprise both heavy and light chains switchclasses during B-cell development. Developing B cells in the bone marrowfirst express membrane bound IgM. During development secretory IgG isexpressed. In the case of antibodies possessing both light and heavychains, a J region is recombined onto a Cμ region to produce an IgMcomprising VH, D and J regions. The IgM producing cell further maturesby switching to a different heavy chain constant region to produce IgAfor example. The mechanism of recombination involves a pseudo lightchain which recombines with the VH part of IgM, the pseudo-light chainbeing present during the to early B cell lineage.

The present inventors realised that understanding the mechanism by whichsingle chain antibodies switch classes and/or undergo affinitymaturation (antibody evolution) during pre B-cell development wouldallow a VHH locus as herein defined to be generated which resulted inthe production of a specific single chain VHH antibody which undergoes aprocess of evolution similar or the same as that of camelid antibodiesproduced in their native environment.

Thus, in a first aspect the present invention provides a method for theproduction of a VHH single heavy chain antibody in a mammal comprisingthe step of expressing a heterologous VHH heavy chain locus in thatmammal.

Preferably the VHH heavy chain locus comprises:

-   -   (a) at least one VHH region each comprising one VHH exon, at        least one D region each comprising one D exon and at least one J        region each comprising one J exon, wherein the VHH region, the D        region and the J region are capable of recombining to form VDJ        coding sequence,    -   (b) a constant heavy chain region comprising at least one Cγ        constant heavy chain gene, and which when expressed does not        express a functional CH1 domain nor a functional CH4 domain,    -   (c) at least one recombination sequence (rss) capable of        recombining a J region of step (a) directly with a Cγ constant        heavy chain gene of step (b).    -   and which locus when expressed is capable of forming a complete        single heavy chain IgG molecule (scIgG).

In a further aspect, the present invention provides a method for theproduction of a camelised VH single heavy chain antibody in a mammalcomprising the step of expressing a camelised VH heavy chain locus inthat mammal

Preferably, the camelised VH heavy chain locus comprises:

-   -   (a) a VH region each comprising one VH exon which is mutated        such that the nucleic acid sequence is the same as a camelid VHH        exon (a ‘camelised VH exon’), a D region comprising one D exon        and a J region comprising one J exon, wherein the VH region, the        D region and the J region are capable of recombining to form VDJ        coding sequence, and    -   (b) a constant heavy chain region comprising at least one Cγ        constant heavy chain gene, and which when expressed does not        express a functional CH1 domain nor a functional CH4 domain,    -   (c) at least one recombination sequence (rss) capable of        recombining a J region of step (a) directly with a Cγ constant        heavy chain gene of step (b).        and which locus when expressed is capable of forming a complete        single heavy chain IgG molecule (scIgG).

The present inventors have shown that in the case of single heavy chainantibodies, class switching occurs to form scAb (a complete single heavychain antibody polypeptide chain). This mechanism involves recombiningthe J region of step (a) directly with a Cγ heavy chain region gene ofthe constant heavy chain region of step (b), preferably in the bonemarrow resulting in the generation of a scIgG (single chain IgGmolecule). The presence of the recombination signal sequence (rss) inthe construct, therefore permits the connection of the J region of step(a) directly to the Cγ gene of step (b).

In the context of the present invention, the mammal is not a human. Thetransgenic mammal is advantageously smaller than a camelid and easier tomaintain and immunise with desired antigens. Ideally, the transgenicmammal is a rodent, such as a rabbit, guinea pig, rat or mouse. Mice areespecially preferred. Alternative mammals, including goats, sheep, cats,dogs and other domestic or wild mammals, may also be employed.

Advantageously heavy chain loci endogenous to the mammal are deleted orsilenced when a single chain antibody is expressed according to themethod of the present invention. Suitable techniques for the later aredescribed in WO00/26373 or WO96/33266 and (L1 and Baker (2000) Genetics156(2): 809-821; Kitamura and Rajewsky (1992); Kitamura and Rajewsky,(1992) Nature 356, 154-156).

The term a ‘VHH single heavy chain antibody’ according to the presentinvention means an antibody molecule which is composed only of heavychains (generally two) and does not comprise any light chains. Eachheavy chain comprises a variable region (encoded by VHH, D and J exons)and a constant region. The constant region further comprises a number ofCH (constant heavy chain domains), advantageously it comprises two: oneCH2 domain and one CH13 domain encoded by a constant region gene. A VHHsingle chain antibody as herein defined does not possess a functionalCH1 domain and also lacks a functional CH4 domain. It is the lack of afunctional CH1 domain (which in conventional antibodies possesses theanchoring place for the constant domain of the light chain) whichaccounts for the inability of the heavy chain antibodies according tothe present invention to associate with light chains to formconventional antibodies.

The term ‘a camelised VH single heavy chain antibody’ according to thepresent invention means an antibody molecule which is composed only ofheavy chains (generally two) and does not comprise any light chains.Each heavy chain comprises a variable region (encoded by ‘a camelised VHexon/s’, D and J exon/s) and a constant region. The constant regioncomprises at least one constant region gene. Each constant region genecomprises a number of constant region exons, each exon encoding aconstant region CH domain. Generally, the constant region comprises twoCH domains: one CH2 domain and one CH3 domain. A camelised VH singlechain antibody as herein defined does not possess a functional CH1domain, in addition it also lacks a functional CH4 domain. It is thelack of a functional CH1 domain (which in conventional antibodiespossesses the anchoring place for the constant domain of the lightchain) which accounts for the inability of the heavy chain antibodiesaccording to the present invention to associate with light chains toform conventional antibodies.

In the context of the present invention, the term ‘heterologous’ means aVHH heavy chain locus as herein described which is not endogenous tothat mammal. That is in the case where the mammal is a camelid i.e. acamel or a llama, then the expression is of a VHH locus which is notnormally found within a camel or llama respectively.

A ‘VHH heavy chain locus’ according to the present invention iscomprised of a ‘VHH region’, a ‘J region’, a ‘D region’ and a ‘constantheavy chain region’. Each VHH region comprises one VHH exon, each Jregion one J exon and each D region one D exon, and each heavy chainconstant region comprises one or more heavy chain constant region genes.In addition each VHH region essentially does not comprise one or morefunctional VH exons.

A ‘VHH exon/region’ in the context of the present invention describes anaturally occurring VHH coding sequence such as those found in Camelidsand any homologue, derivative or fragment thereof as long as theresultant exon/region recombine with a D exon/region, a J exon/regionand a constant heavy chain region (which comprises several exons)according to the present invention to generate a VHH single chainantibody as herein defined, when the nucleic acid is expressed.

A ‘camelised VH heavy chain locus’ according to the present invention iscomprised of a ‘a camelised VH region’ as herein defined, a ‘J region’,a ‘D region’ and a ‘constant heavy chain region’. Each camelised VHregion comprises one camelised VH exon, each J region one J exon andeach D region one D exon and each heavy chain constant region comprisesone or more heavy chain constant region exons.

A ‘camelised VH exon/region’ in the context of the present inventiondescribes a naturally occurring VH coding sequence derived from mammalsother than Camelids for example a human which has been mutated such thatthe sequence is the same as that of a Camelid exon. A camelised VH exonaccording to the present invention also includes within its scope anyhomologue, derivative or fragment of the exon as long as the exon/regioncan recombine with a D region/exon, a J region/exon and a constant heavychain region comprising one or more exons according to the presentinvention to generate a camelised VH single chain antibody as hereindefined.

VHH and VH exons may be derived from naturally occurring sources or theymay be synthesised using methods familiar to those skilled in the artand described herein.

Likewise in the context of the present invention the terms ‘a D exon’and ‘a J exon’ include naturally occurring sequences of D and J exonswhich are found in Camelids or other species of mammals. The terms Dexon and J exon also include within their scope derivatives, homologuesand fragments thereof as the resultant exon can recombine with theremaining components of a heavy chain antibody locus as herein described(either camelised VH or VHH) to generate a single chain antibody asherein described. D and J exons/regions may be derived from naturallyoccurring sources or they may be synthesised using methods familiar tothose skilled in the art and described herein.

In addition, a heavy chain antibody locus according to the presentinvention (either VHH or camelised VH) comprises a region of DNAencoding a constant heavy chain polypeptide (a constant heavy chainregion).

Each constant heavy chain region essentially comprises at least oneconstant region heavy chain gene which is Cγ, so that generation ofsingle chain IgG can occur. Each constant heavy chain gene comprises oneor more constant heavy chain exons which may be of Camelid ornon-Camelid origin and are selected from the group consisting of Cδ,Cγ₁₋₄, Cε and Cα₁₋₂. Preferably, at least one heavy chain constantregion exon in a heavy chain antibody locus according to the presentinvention is of human, mouse or rabbit origin. Advantageously, at leastone Cγ heavy chain exon is of human origin. When expressed the constantheavy chain region lacks a functional CH1 and CH4 domain which arepresent in dual chain antibodies. Advantageously, only one or more Cγ₂and/or Cγ₃ genes with modified (non-functional) CH1 domains are presentin the constant heavy chain region of the present invention.

A ‘constant heavy chain region exon’ (‘C_(H) exon’) as herein definedincludes the sequences of naturally occurring C_(H) exons such as thosefound in camelids or humans or other mammals including rabbits and mice.The term ‘C_(H) exon’ also includes within its scope derivatives,homologues and fragments thereof in so far as the C_(H) exon is able toform a functional single heavy chain antibody (comprising either regionsencoded by VHH exons or camelised VH exons) as herein defined when it isa component of a constant heavy chain region.

Generally, C_(H) genes comprise three or four exons (C_(H)1-C_(H)4) thatencode different domains of each constant heavy chain polypeptide, withgenerally two polypeptides constituting a single heavy chain antibody asherein described. However, as discussed previously, VHH and camelised VHsingle chain antibodies do not possess an functional CH1 (containing thelight chain domain anchoring region) or CH4. Thus, single heavy chainantibody loci according to the present invention possess one or moregenes which do not express functional CH1 or CH4 domains. This may occurby mutation, deletion substituted or other treatment of the CH1 and CH4exons of the constant heavy region gene.

In a preferred embodiment of the invention a single chain VHH locuscomprises at least one constant heavy chain gene wherein the nucleicacid encoding the CH1 and the CH4 domain is mutated, deleted orsubstituted or otherwise treated so that the constant heavy chain ofexpressed VHH single chain antibodies as herein defined does not containa functional CH1 domain and a CH4 domain.

For the avoidance of doubt, the term ‘rabbit origin’ or ‘human origin’as referred to above, means that the nucleic acid sequence of one ormore exons comprising a heavy chain antibody locus (either camelised VHor VHH) according to the present invention is the same as one or morenaturally occurring rabbit or human antibody locus exons. One skilled inthe art will appreciate that these exons may be derived from naturalsources or may be synthesised using methods familiar to those skilled inthe art and described herein.

Each VHH or ‘camelised VH region’ comprises one VHH exon or ‘camelisedVH exon’ respectively. Each J region and D region comprises one J and Dexon respectively. Preferably, each heavy chain locus comprises morethan one, more than 2, more than 3, more than 4, more than 5, more than6 J and/or D regions/exons. Most preferably, a VHH locus or camelised VHlocus according to the present invention comprises the same number ofVHH exons/regions and/or D exons/regions and/or J exons/regions as thosefound in a Camelid.

Advantageously, the method of this aspect of the present invention isfor the production of a single chain antibody by the expression of a VHHheavy chain locus or camelised VH heavy chain locus comprising one ormore constant heavy chain exons of human, rabbit or mouse origin asherein defined. That is, preferably a single heavy chain antibody of thepresent invention is generated by the expression of a hybridcamelid/human locus or a hybrid camelid/rabbit locus or a hybridcamelid/mouse locus. In an especially preferred embodiment of thisaspect of the invention, the single heavy chain locus expressedaccording to the method of the present invention comprises all VHH exonsof Camelid origin and all D, J and constant heavy chain region exons ofhuman origin, rabbit or mouse origin. In a further preferred embodimentof this aspect of the invention, the single heavy chain locus expressedaccording to the method of the present invention comprises all camelisedVH exons and all D, J and constant heavy chain region exons of humanorigin, or rabbit or mouse origin.

In a preferred embodiments of the above aspects of the invention, theheavy chain locus further comprises one or more cassette sites enablingthe direct cassetting of the locus from one vector to another.Advantageously, one or more cassette sites are located in the 5′ leadersequence of the locus and/or the 3′ untranslated region of the locus.Preferably, one or more cassettes sites are located in both the 5′leader sequence of the locus and the 3′ untranslated region of thelocus. The direct cassetting permits, for example, movement of nucleicacid into a bacterial expression vector for the addition of tags,signals and the like.

This approach of generating hybrid single heavy chain antibodies asdescribed above maybe of particular use in the generation of antibodiesfor human therapeutic use as often the administration of antibodies to aspecies of vertebrate which is of different origin from the source ofthe antibodies results in the onset of an immune response against thoseadministered antibodies. Hybrid camelid/human single chain antibodiesare therefore potentially less immunogenic than Camelid single chainantibodies when administered to a human.

In the context of the present invention, the same includes substantiallythe same. Substantially the same means greater than 80% homologous,preferably greater than 85%, 90%, 95% homologous. More preferablygreater than 96, 97, 98% homologous. Most preferably, substantially thesame means that the mutated human VH region is greater than 99%homologous with a Camelid VHH region

In a further aspect, the present invention provides a VHH single heavychain antibody obtainable according to the method of the presentinvention wherein that part of the antibody encoded by a VHH exon isencoded by an exon of camelid origin and the remainder of the antibodymolecule is encoded by exons of human origin.

In yet a further aspect, the present invention provides a VHH singleheavy chain antibody obtainable according to the method of the presentinvention, wherein that part of the antibody encoded by a VHH exon isencoded by an exon of camelid origin and the constant heavy chain regionis encoded by one or more exon/s of rabbit origin.

In a further aspect still, the present invention provides a VHH singleheavy chain antibody obtainable according to the method of the presentinvention wherein that part of the antibody encoded by a VHH exon isencoded by an exon of camelid origin and the constant heavy chain regionis encoded by one or more exon/s of mouse origin.

In yet a further aspect, the present invention provides a camelisedsingle heavy chain antibody obtainable by the method of the presentinvention

Advantageously, a camelised VH single heavy chain antibody according tothis aspect of the present invention is entirely encoded by exons ofhuman origin as herein defined.

In a further preferred embodiment of this aspect of the invention, acamelised VH single heavy chain antibody comprises a constant heavychain region encoded by one or more exon/s of rabbit origin.

In a further embodiment still, a camelised VH single heavy chainantibody according to this aspect of the invention comprises a constantheavy chain region encoded by one or more exon/s of mouse origin.

Antibodies produced according to the method of the present inventionhave the advantage over those of the prior art in that they undergo aprocess of class switching which is similar or the same as that of asingle chain Camelid antibody generated in its normal environment.Antibodies obtainable according to the methods of the present inventionmay be monoclonal or polyclonal antibodies. Advantageously, they aremonoclonal antibodies. Antibodies may be generated using methods knownto those skilled in the art. Advantageously hybridomas may be used forgenerating monocloanl antibodies. Techniques will be familiar to thoseskilled in the art and are described herein.

In yet a further aspect, the present invention provides a vectorcomprising a VHH heavy chain locus according to the present invention.

In a further aspect still, the present invention provides a vectorcomprising a camelised VH heavy chain locus according to the presentinvention.

Suitable vectors will be familiar to those skilled in the art.Advantageously, a vector suitable of inserting large amounts of nucleicacid, sufficient to encode an entire immunoglobulin heavy chain locusare preferred. Suitable vectors include yeast and bacterial artificialchromosomes such as YACs and BACs. Advantageously, the vectors areconstructed so that direct cassetting of nucleic acid encoding a singleheavy chain antibody locus as herein defined into a different vector canbe performed. For example the reverse transcribed cDNA coding for asingle heavy chain antibody may be ‘cassetted’ into a bacterialexpression vector allowing for the addition of tags, signals or epitopesand the like.

In yet a further aspect, the present invention provides a host celltransformed with a VHH locus according to the present invention.

In a further aspect still, the present invention provides a transgenicmammal expressing a heterologous VHH heavy chain locus according to thepresent invention.

In yet a further aspect, the present invention provides a transgenicmammal expressing a camelised VH heavy chain locus according to thepresent invention.

In the context of the present invention, the term ‘a transgenic mammal’does not include within its scope a transgenic human. Preferably atransgenic mammal according to the present invention is smaller than aCamelid. Preferably it is selected from the group consisting of a mouse,rat, guinea-pig, hamster, monkey and rabbit. Advantageously, it is amouse.

Advantageously heavy chain loci endogenous to the transgenic animal aredeleted or silenced in a transgenic mammal according to the presentinvention. Suitable techniques for the later are described in WO00/26373or WO96/33266 and (L1 and Baker (2000) Genetics 156(2): 809-821;Kitamura and Rajewsky (1992); Kitamura and Rajewsky, (1992) Nature 356,154-156).

Antibody producing cells may be derived from transgenic animalsaccording to the present invention and used for example in thepreparation of hybridomas for the production of VHH single chainantibodies as herein defined. In addition or alternatively, nucleic acidsequences may be isolated from transgenic mammals according to thepresent invention and used to produce single chain antibodies, usingrecombinant DNA techniques which are familiar to those skilled in theart. Alternatively or in addition, specific single chain antibodies maybe generated by immunising a transgenic animal according to the presentinvention.

Thus in a further aspect, the present invention provides a method forthe production of single chain antibodies by immunising a transgenicmammal according to the present invention with an antigen.

In a preferred embodiment of this aspect of the invention, the mammal isa mouse.

In the context of the present invention, the term ‘immunising’ a mammalmeans administering to a transgenic mammal of the present invention anantigen such that an immune response is elicted against that antigen.Suitable methods for the immunisation of mammals will be familiar tothose skilled in the art and are described herein. Suitable antigens maybe naturally occurring or synthetic. Naturally occurring antigensinclude proteins which may be for example enzymes or cofactors, peptidesand nucleic acid molecules. One skilled in the art will appreciate thatthis list is not intended to be exhaustive.

In a further aspect, the present invention provides the use of a singleheavy chain antibody as herein described as an intracellular bindingreagent.

In a further aspect, the present invention provides, the use of a singlechain antibody according to the present invention as an enzymeinhibitor.

In a further aspect still, the present invention provides the use of anantibody obtainable by the method of the present invention in thepreparation of a medicament for the prophylaxis and/or treatment ofdisease.

In a final aspect, the present invention provides the use of a heavychain antibody locus according to the present invention in theprophylaxis or treatment of disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a preferred single chain antibody locus according to thepresent invention.

DEFINITIONS

‘A gene’ comprises one or more exons coding for a complete mRNA. An‘antibody gene’ comprises V, D, J exons which recombine to form a VDJcoding region and which then further recombine with a constant heavychain region comprising one or more constant heavy chain exons. Thereare many sub-groups of V, D J and C exons. One particular V region hasone exon, one D region has one exon, one J region has one exon and one Cregion has several exons. Together they from a complete gene afterrecombination when one V exon, one D exon, one J exon and one C regionhave been selected.

‘Exon’ and ‘intron’. An Exon is a coding or messenger sequence ofdeoxynucleotides. That is, it is any sequence of DNA in eukaryotes thatwill be ultimately expressed in mature mRNA or rRNA molecules. Exons arecommonly interspersed with introns. Introns are non-coding DNAsequences. That is they are DNA sequences which are not ultimatelyexpressed in a mature RNA molecule.

Introns are spliced out from newly transcribed RNA to order to generatemature mRNA.

A ‘VHH heavy chain locus’ according to the present invention iscomprised of a ‘VHH region’, a ‘J region/exon’, a ‘D region/exon’ and a‘constant heavy chain region’. Each VHH region comprises one VHH exon,each J region one J exon and each D region one D exon and each heavychain constant region comprises one or more heavy chain constant regionexons.

A ‘VHH exon’ in the context of the present invention describes anaturally occurring VHH coding sequence such as those found in Camelidsand any homologue, derivitive or fragment thereof as long as theresultant exon can when a constituent of a VHH region as herein definedrecombine with at least one D region, at least one J region and at leastone constant heavy chain region according to the present invention togenerate a VHH single chain antibody as herein defined, when the nucleicacid is expressed.

A ‘camelised VH heavy chain locus’ according to the present invention iscomprised of one or more ‘camelised VH region/s’ as herein defined, oneor more ‘J region/s’, one or more ‘D region/s’ and a ‘constant heavychain region’. Each camelised VH region comprises one camelised VH exon,each J region one J exon and each D region one D exon and each heavychain constant region comprises one or more heavy chain constant regiongenes.

A ‘camelised VH exon’ in the context of the present invention describesa naturally occurring VH coding sequence derived from mammals other thanCamelids for example a human which has been mutated such that thesequence is the same as that of a Camelid exon. A camelised VH exonaccording to the present invention also includes within its scope anyhomologue, derivative or fragment of the exon as long as the resultantexon can, when a constituent of a camelised VH region as herein definedrecombine with at least one D region, one J region and one constantheavy chain region according to the present invention to generate acamelised VH single chain antibody as herein defined.

A ‘constant heavy chain region exon’ (‘C_(H) exon’) as herein definedincludes the sequences of naturally occurring C_(H) exons such as thosefound in camelids or humans or other mammals including rabbits and mice.The term ‘C_(H) exon’ also includes within its scope derivatives,homologues and fragments thereof in so far as the C_(H) exon is able toform a functional single heavy chain antibody as herein defined when itis a component of a constant heavy chain region. Generally, C_(H) exonsare of four different types (C_(H)1-C_(H)4) that encode differentportions (domains) of each constant heavy chain polypeptide. However,VHH and camelised VH single chain antibodies according to the presentinvention do not possess a functional CH1 domain (containing the lightchain domain anchoring region) nor do they possess a functional CH4domain. There are a number of sub-groups of constant heavy chain regionexons. Different antibody classes possess different CH exons forinstance, IgM molecules possess one or more Cμ constant region exons andIgG molecules possess one or more Cγ exons.

The term a ‘VHH single heavy chain antibody’ according to the presentinvention means an antibody molecule which is composed only of heavychains (generally two) and does not comprise any light chains. Eachheavy chain comprises a variable region (encoded by VHH, D and J exons)and a constant region. The constant region further comprises a number ofCH domains encoded by constant heavy region exons, generally itcomprises two: one CH2 domain and one CH3 domain. A VHH single chainantibody as herein defined does not possess a functional CH1 domain nora functional CH4 domain. It is the lack of a functional CH1 domain(which in conventional antibodies possesses the anchoring place for theconstant domain of the light chain) which accounts for the inability ofthe heavy chain antibodies according to the present invention toassociate with light chains to form conventional antibodies. Thesub-class of antibodies known as scIgG2 and/or scIgG3 comprise only Cγ₂and/or Cγ₃ genes.

The term ‘a camelised VH single heavy chain antibody’ according to thepresent invention means an antibody molecule which is composed only ofheavy chains (generally two) and does not comprise any light chains.Each heavy chain comprises a variable region (encoded by ‘a camelised VHexon’, D and J exon/s) and a constant region. The constant regionencoded by constant region exons further encodes a number of CH domains,generally it comprises two: one CH2 domain and one CH3 domain. Acamelised VH single chain antibody as herein defined does not possess afunctional CH1 domain or a functional CH4 domain. It is the lack of afunctional domain (which in conventional antibodies possesses theanchoring place for the constant domain of the light chain) whichaccounts for the inability of the heavy chain antibodies according tothe present invention to associate with light chains to formconventional antibodies.

‘Antibodies’ as used herein, refers to antibodies or antibody fragmentscapable of binding to a selected target, and includes monoclonal andpolyclonal antibodies, engineered antibodies including chimeric,CDR-grafted and humanised antibodies, and artificially selectedantibodies produced using phage display or alternative techniques. Smallantibody fragments possess advantageous properties for diagnostic andtherapeutic applications on account of their small size and consequentsuperior tissue distribution.

‘Antibody evolution’ describes the process of class switching andaffinity maturation (somatic hypermutation) which occurs during antibodydevelopment and which results in the generation of antibodies which bindselectively and with high affinity.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and

Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th) Ed,John Wiley &. Sons, Inc. which are incorporated herein by reference) andchemical methods. In addition Harlow & Lane., A Laboratory Manual ColdSpring Harbor, N.Y., is referred to for standard ImmunologicalTechniques.

VH/h Heavy Chain Loci of the Present Invention

In a first aspect, the present invention provides a method for theproduction of a VHH single heavy chain antibody in a mammal comprisingthe step of expressing a to heterologous VHH heavy chain locus in thatmammal.

In a further aspect, the present invention provides a method for theproduction of a camelised VH single heavy chain antibody in a mammalcomprising the step of expressing a camelised VH heavy chain locus inthat mammal.

The construction of the various VHH heavy chain loci according to thepresent invention are as described in the summary of the invention.

Advantageously, a locus of the invention comprises one or more FRT (flprecombination target) sites (http://www.esb.utexus.edu), and two or moreLoxP sites (which consists of two thirteen by inverted repeats separatedby an 8 bp asymmetric spacer region (Brian Sauer, Methods of Enzymology;1993, Vol 225, 890-900).

Preferably, there are at least two loxP sites in a locus according tothe present invention. The presence of the FRT site/s in the locusallows the production of single copy transgenics, whilst the presence ofthe Lox sites allows the deletion of IgM and IgD heavy chain genes ifrequired.

(A) Vectors

The present invention also provides vectors including a construct of thepresent invention. Essentially two types of vectors are provided,replication vectors and transformation vectors.

(I) Replication Vectors

Constructs of the invention can be incorporated into a recombinantreplicable vector such as a BAC vector. The vector may be used toreplicate the construct in a compatible host cell. Thus, in a furtherembodiment, the invention provides a method of making constructs of theinvention by introducing a construct of the invention into a replicablevector, introducing the vector into a compatible host cell, and growingthe host cell under conditions which bring about replication of theconstruct. The construct may be recovered from the host cell. Suitablehost cells include bacteria such as E. coli, yeast, mammalian cell linesand other eukaryotic cell lines, for example insect Sf9 cells(baculovirus).

(III) Transformation Vectors

The constructs of the present invention may also be incorporated into avector capable of inserting the construct into a recipient genome andthus achieving transformation. In addition to the construct of thepresent invention such transformation vectors may include one or more ofthe following components.

Promoters

The promoter is typically selected from promoters which are functionalin mammalian cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression is to occur. With respect to eukaryotic promoters, theymay be promoters that function in a ubiquitous manner (such as promotersof alpha-actin, beta-actin, tubulin) or, alternatively, atissue-specific manner (such as promoters of immunoglobulin genes). Theymay also be promoters that respond to specific stimuli, for examplepromoters that bind steroid hormone receptors. Viral promoters may alsobe used, for example the Moloney murine leukaemia virus long terminalrepeat (MMLV LTR) promoter, the Rous sarcoma virus (RSV) LTR promoter orthe human cytomegalovirus (CMV) IE promoter. It may also be advantageousfor the promoters to be inducible so that the levels of expression ofthe heterologous gene can be regulated during the life-time of the cell.Inducible means that the levels of expression obtained using thepromoter can be regulated.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences.Tissue-specific enhancers capable of regulating expression inantibody-producing cells are preferred. In particular, the heavy-chainenhancer required for the successful activation of the antibody genelocus in vivo (Serwe, M., and Sablitzky, F., EMBO J. 12, p2321-2321,1993) may be included. Locus control regions (LCRs), particularly theimmunoglobulin LCR, may also be used. Chimeric promoters may also beused comprising sequence elements from two or more different promoters.

Other Vector Components

In addition to a promoter and the construct, vectors of the presentinvention preferably contain other elements useful for optimalfunctioning of the vector in the mammal into which the vector isinserted. These elements are well known to those of ordinary skill inthe art, and are described, for example in Sambrook et al., MolecularCloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, 1989.

Construction of Vectors

Vectors used for transforming mammalian embryos are constructed usingmethods well known in the art, including, without limitation, thestandard techniques of restriction endonuclease digestion, ligation,plasmid and DNA and RNA purification, DNA sequencing, and the like asdescribed, for example in Sambrook, Fritsch, and Maniatis, eds.,Molecular Cloning: A Laboratory Manual., (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. [1989]). In general, vector constructionwill include the following steps:

a) The endogenous mouse locus is inactivated, for example using one ofthe published knockout procedures (e.g. Kitamara, D and Rajewski K.,Nature 352, p154-156, 1992).b) The DJ and IgM region of a suitable heavy chain region as hereindescribed is localised as a recombinant DNA from a human PAC, BAC or YAClibrary and cloned as a restriction enzyme fragment, for instance a Sal1fragment. This region also contains the heavy chain enhancer requiredfor the successful activation of the antibody gene locus in vivo (seeSerwe, M., Sablitzky, F., EMBO J. 12, p2321-2321, 1993).c) A number of VHH or ‘camelised VH exons’ are first cloned as cosmidsthrough the construction of a suitable genomic DNA library byconventional techniques. Since the VHH exons are located among VH exonsas herein described they are subsequently cloned along with the VHHexons. Thus an array of VH and VHH exons is made. This array of genescan be isolated as a MluI (or other restriction enzyme) fragment.d) The 3′ human immunoglobulin heavy chain LCR, a regulatory regionrequired for the expression of the locus, is cloned as an SceIrestriction fragment.e) The constant region heavy chain exons are cloned as a separaterestriction fragment. The C_(H)1 and/or C_(H)4 domains encoded by theirrespective exons are rendered non-functional by homologous recombinationin bacteria (Imam et al., 2000) by removing the splice acceptorsequences of the C_(H)1 exon and/or C_(H)4 exon (Nguyen et al., ibid,).

Steps b-e provide the pieces for a ‘VHH heavy chain locus’ or ‘acamelised VH heavy chain locus’ (FIG. 3) that should take over thefunction of the inactivated mouse locus described under a). These lociare constructed by cloning each of the fragments in the appropriateorder into a suitable vector, for example a BAC vector containing alinker region with all of the restriction sites described above (FIG.1). Loci created according to the method of the present invention aregenerally in the order of 200-250 kB in size. They can be isolated andpurified away from the vector by standard laboratory techniques whichwill be familiar to those skilled in the art. The purified nucleic acidencoding the ‘VHH heavy chain locus’ or ‘a camelised VH heavy chainlocus’ according to the present invention (FIG. 3) may be subsequentlyintroduced into fertilized mouse eggs derived from the knock-out micedescribed in a) by standard techniques to obtain transgenic miceexpressing one or more loci according to the present invention.

Single Chain Antibodies According to the Present Invention

It will be understood that term ‘a single heavy chain antibody’ and ‘VHHheavy chain loci’ according to the present invention also includehomologous polypeptide and nucleic acid sequences obtained from anysource, for example related cellular homologues, homologues from otherspecies and variants or derivatives thereof.

Thus, the present invention encompasses variants, homologues orderivatives of the single heavy chain antibodies and VHH heavy chainloci as herein described.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 80, 85, 90, 95, 96,97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9% identical, preferably at least98 or 99% identical at the amino acid level over at least 30, preferably50, 70, 90 or 100 amino acids. Although homology can also be consideredin terms of similarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without to penalizing unduly the overall homology score. Thisis achieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Methods for the Production of Single Chain Antibodies According to thePresent Invention (A) Transgenic Animals

The loci and vectors of the present invention may be introduced into ananimal to produce a transgenic animal. Thus, the present invention alsoprovides a transgenic animal including a construct described herein.

Inserting the loci into the genome of a recipient animal may be achievedusing any technique apparent to those skilled in the art, for example,microinjection. Following introduction of nucleic acid into a fertilizedegg, reimplantation is accomplished using standard methods which will befamiliar to those skilled in the art. Usually, the surrogate host isanaesthetized, and the eggs are inserted into the oviduct. The number ofeggs implanted into a particular host will vary, but will usually becomparable to the number of offspring the species naturally produces.

Alternatively, the DNA may be introduced into embryonic stem cells (ES)cells which can be inserted into a host embryo to derive transgenic miceby standard technology.

In a further embodiment the DNA can be introduced into any cell. Thenuclei of these cells are used to replace the nucleus of a fertilisedegg which may be of any species to give rise to transgenic animals. Thistechnique of nuclear transfer is familiar to those skilled in the art.

Transgenic offspring of the surrogate host may be screened for thepresence of the transgene by any suitable method. Screening is oftenaccomplished by Southern or Northern analysis, using a probe that iscomplementary to at least a portion of the transgene. Western blotanalysis using a ligand specific for the antibody encoded by thetransgene may be employed as an alternative or additional method forscreening. Typically, the tissues or cells believed to express thetransgene at the highest levels are tested, although any tissues or celltypes may be used for this analysis.

Progeny of the transgenic mammals may be obtained by mating thetransgenic mammal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic mammal. Where in vitrofertilization is used, the fertilized embryo may be implanted into asurrogate host or incubated in vitro, or both. Where mating is used toproduce transgenic progeny, the transgenic mammal may be backcrossed toa parental line. Using either method, the progeny may be evaluated forthe presence of the transgene using methods described above, or otherappropriate methods.

The animal may be varied provided it is a mammal. Preferably, the animalis a non-human mammal such as a rodent and even more preferably a rat ormouse. In this regard, it is also preferred that the recipient animal isincapable of producing antibodies that include light chains or at thevery least has a reduced capacity to produce such antibodies. To achievethis end the recipient animal may be a “knock out” animal that iscapable of having one or more of the genes required for the productionof antibodies with light chains turned off or suppressed.

By using recipient animals incapable of producing antibodies thatinclude light chains or at the very least with only a reduced capacityto produce such antibodies, the method of the present invention enablesthe efficient production of large quantities of single chain antibodiesand antibody producing cells from a transgenic animal according to thepresent invention upon challenge with a given antigen.

(B) Phage Display Technology

Vectors for phage display fuse the encoded polypeptide to, e.g., thegene III protein (pIII) or gene VIII protein (pVIII) for display on thesurface of filamentous phage, such as M13. See Barbas et al., PhageDisplay: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001)(ISBN 0-87969-546-3); Kay et al. (eds.), Phage Display of Peptides andProteins: A Laboratory Manual, San Diego: Academic Press, Inc., 1996;Abelson et al. (eds.), Combinatorial Chemistry, Methods in Enzymologyvol. 267, Academic Press (May 1996).

Prokaryotic hosts are particularly useful for producing phage displayedantibodies of the present invention. The technology of phage-displayedantibodies, in which antibody variable region fragments are fused, forexample, to the gene III protein (pIII) or gene VIII protein (pVIII) fordisplay on the surface of filamentous phage, such as M13, is by nowwell-established, Sidhu, Curr. Opin. Biotechnol. 11(6):610-6 (2000);Griffiths et al., Curr. Opin. Biotechnol. 9(1):102-8 (1998); Hoogenboomet al., Immunotechnology, 4(1):1-20 (1998); Rader et al., CurrentOpinion in Biotechnology 8:503-508 (1997); Aujame et al., HumanAntibodies 8:155-168 (1997); Hoogenboom, Trends in Biotechnol. 15:62-70(1997); de Kruif et al., 17:453-455 (1996); Barbas et al., Trends inBiotechnol. 14:230-234 (1996); Winter et al., Ann. Rev. Immunol. 433-455(1994), and techniques and protocols required to generate, propagate,screen (pan), and use the antibody fragments from such libraries haverecently been compiled, Barbas et al., Phage Display: A LaboratoryManual, Cold Spring Harbor Laboratory Press (2001) (ISBN 0-87969-546-3);Kay et al. (eds.), Phage Display of Peptides and Proteins: A LaboratoryManual, Academic Press, Inc. (1996); Abelson et al. (eds.),Combinatorial Chemistry, Methods in Enzymology vol. 267, Academic Press(May 1996), the disclosures of which are incorporated herein byreference in their entireties. For the phage display of antibodies asherein described including fragments thereof, advantageously, they arefused to the phage g3p protein.

(C) Hybridomas

Recombinant DNA technology may be used to produce single chainantibodies according to the present invention using an establishedprocedure, in bacterial or preferably mammalian cell culture. Theselected cell culture system preferably secretes the single chainantibody product.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which are the customary standardculture media, for example Dulbecco's Modified Eagle Medium (DMEM) orRPMI 1640 medium, optionally replenished by a mammalian serum, e.g.foetal calf serum, or trace elements and growth sustaining supplements,e.g. feeder cells such as normal mouse peritoneal exudate cells, spleencells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin,low density lipoprotein, oleic acid, or the like. Multiplication of hostcells which are bacterial cells or yeast cells is likewise carried outin suitable culture media known in the art, for example for bacteria inmedium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, or M9Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, orComplete Minimal Dropout Medium.

In vitro production provides relatively pure immunoglobulin preparationsand allows scale-up to give large amounts of the desiredimmunoglobulins. Techniques for bacterial cell, yeast or mammalian cellcultivation are known in the art and include homogeneous suspensionculture, e.g. in an airlift reactor or in a continuous stirrer reactor,or immobilised or entrapped cell culture, e.g. in hollow fibres,microcapsules, on agarose microbeads or ceramic cartridges.

Large quantities of the desired immunoglobulins can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired immunoglobulins are injected into histocompatiblemammals to cause growth of antibody-producing tumours. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the immunoglobulins are isolated from the body fluids ofthose mammals. For example, hybridoma cells obtained by fusion ofsuitable myeloma cells with antibody-producing spleen cells from Balb/cmice, or transfected cells derived from hybridoma cell line Sp2/0 thatproduce the desired antibodies are injected intraperitoneally intoBalb/c mice optionally pre-treated with pristane, and, after one to twoweeks, ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, incorporated herein by reference. Techniques for the preparationof recombinant antibody molecules is described in the above referencesand also in, for example, EP 0623679; EP 0368684 and EP 0436597, whichare incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of cells expressing thedesired target by immunoblotting, by an enzyme immunoassay, e.g. asandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, those present in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulphate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-)affinity chromatography, e.g. affinitychromatography with the target molecule or with Protein-A.

(3) Immunisation of a Transgenic Animal

In a further aspect, the present invention provides a method for theproduction of single chain antibodies according to the present inventioncomprising administering an antigen to a transgenic animal according tothe present invention.

The single chain antibodies produced from transgenic animals of thepresent invention include polyclonal and monoclonal single chainantibodies and fragments thereof. If polyclonal antibodies are desired,the transgenic animal (e.g., mouse, rabbit, goat, horse, etc.) may beimmunised with an antigen and serum from the immunised animal collectedand treated according to known procedures. If serum containingpolyclonal antibodies contains antibodies to other antigens, thepolyclonal antibodies of interest can be purified by immunoaffinitychromatography and such like techniques which will be familiar to thoseskilled in the art. Techniques for producing and processing polyclonalantisera are also known in the art.

Uses of Single Chain Antibodies According to the Present Invention

Single chain antibodies including fragments thereof according to thepresent invention may be employed in in vivo therapeutic andprophylactic applications, in vitro and in vivo diagnostic applications,in vitro assay and reagent applications, and the like.

Therapeutic and prophylactic uses of single chain antibodies accordingto the invention involve the administration of the above to a recipientmammal, such as a human.

‘Camelised VH single chain heavy chain antibodies’ possess severaladvantages over camelid VHH single chain antibody molecules in thetreatment of humans. For example camelised VH1 single chain antibodiespossess a protein A binding site in the case of antibodies based on theVH3 gene family. In addition, camelised VH single chain antibodies areexpected to show lower immunogenicity than camelid VHH single chainantibodies when administered to humans.

It will also be appreciated that ‘camelised VH single heavy chainantibodies’ and ‘camelid VHH single heavy chain antibodies’ have somedifferent physical characteristics than conventional dual chainantibodies. For example, due to the lack of a functional CH1 heavydomain, antibodies of the present invention do not bind complementmolecule C1 q which is involved in activation of the classical pathwayof complement.

Substantially pure single chain antibodies including fragments thereofof at least 90 to 95% homogeneity are preferred for administration to amammal, and 98 to 99% or more homogeneity is most preferred forpharmaceutical uses, especially when the mammal is a human. Oncepurified, partially or to homogeneity as desired, the single chainantibodies as herein described may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures using methods known to those skilled in theart.

The selected single chain antibodies of the present invention willtypically find use in preventing, suppressing or treating inflammatorystates, allergic hypersensitivity, cancer, bacterial or viral infection,and autoimmune disorders (which include, but are not limited to, Type Idiabetes, multiple sclerosis, rheumatoid arthritis, systemic lupuserythematosus, Crohn's disease and myasthenia gravis), and in preventingtransplant rejection. For instance, depletion of the regulatory T cellsor interference with their recruitment may result in an enhanced immuneresponse which may be of particular use in the treatment of infectionswhich otherwise escape a normal immune response.

In addition, the selected single chain antibodies including fragmentsthereof maybe useful for modulating an immune response in regions of avertebrate where they are not normally located. For example, one or moreantibodies used as herein described may be perfused, injected, into atissue of a vertebrate, using techniques known to those skilled in theart. The presence of an antibody as described herein, in such an ectopicenvironment may be useful in the modulation of an immune response duringfor example, transplant rejection and the like.

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe selected antibodies of the present invention in protecting againstor treating the disease are available. Methods for the testing ofsystemic lupus erythematosus (SLE) in susceptible mice are known in theart (Knight et al. (1978) J. Exp. Med., 147: 1653; Reinersten et al.(1978) New Eng. J. Med., 299: 515). Myasthenia Gravis (MG) is tested inSJL/J female mice by inducing the disease with soluble AchR protein fromanother species (Lindstrom et al. (1988) Adv. Immunol., 42: 233).Arthritis is induced in a susceptible strain of mice by injection ofType II collagen (Stuart et al. (1984) Ann. Rev, Immunol., 42: 233). Amodel by which adjuvant arthritis is induced in susceptible rats byinjection of mycobacterial heat shock protein has been described (VanEden et al. (1988) Nature, 331: 171). Thyroiditis is induced in mice byadministration of thyroglobulin as described (Maron et al. (1980) J.Exp. Med., 152: 1115). Insulin dependent diabetes mellitus (IDDM) occursnaturally or can be induced in certain strains of mice such as thosedescribed by Kanasawa et al. (1984) Diabetologia, 27: 113. EAE in mouseand rat serves as a model for MS in human. In this model, thedemyelinating disease is induced by administration of myelin basicprotein (see Paterson (1986) Textbook of Immunopathology, Mischer etal., eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al.(1973) Science, 179: 478: and Satoh et al. (1987) J. Immunol., 138:179).

Generally, the selected single chain antibodies of the present inventionwill be utilised in purified form together with pharmacologicallyappropriate carriers. Typically, these carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, any includingsaline and/or buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride andlactated Ringer's. Suitable physiologically-acceptable adjuvants, ifnecessary to keep a polypeptide complex in suspension, may be chosenfrom thickeners such as carboxymethylcellulose, polyvinylpyrrolidone,gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The selected single chain antibodies including fragments thereof, of thepresent invention may be used as separately administered compositions orin conjunction with other agents. These can include variousimmunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycinor cisplatinum, and immunotoxins. Pharmaceutical compositions caninclude “cocktails” of various cytotoxic or other agents in conjunctionwith the selected antibodies, or T-cells of the present invention oreven combinations of the selected antibodies according to the presentinvention.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected antibodies, receptors or binding proteinsthereof of the invention can be administered to any patient inaccordance with standard techniques. The administration can be by anyappropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counterindications and other parameters to be taken into account by theclinician.

The selected antibodies, of this invention can be lyophilised forstorage and reconstituted in a suitable carrier prior to use. Knownlyophilisation and reconstitution techniques can be employed. It will beappreciated by those skilled in the art that lyophilisation andreconstitution can lead to varying degrees of functional activity lossand that use levels may have to be adjusted upward to compensate.

In addition, antibodies according to the present invention may be usedfor diagnostic purposes. For example antibodies as herein described maybe generated or raised against antigens which are specifically expressedduring disease states or whose levels change during a given diseasestates.

For certain purposes such as diagnostic or tracing purposes labels maybe added. Suitable labels include but are not limited to any of thefollowing, radioactive labels, NMR spin labels and fluorescent labels.Means for the detection of the labels will be familiar to those skilledin the art.

Examples of suitable radioactive labels include technetium 99m(^(99m)Tc) or iodine-123 (¹²³I) Labels such as iodine-123, iodine-313,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron allow detection of the label using NMR. Labels such as11C methionine and FDG are suitable for use in the technique of positronemission tomography. Descriptions of procedures and protocols for usingPET are familiar to those skilled in the art.

A suitable fluorophore is GFP or a mutant thereof. GFP and its mutantsmay be synthesised together with the antibodies of the present inventionor target molecule by expression therewith as a fusion polypeptide,according to methods well known in the art. For example, a transcriptionunit may be constructed as an in-frame fusion of the desired GFP and theimmunoglobulin or target, and inserted into a vector as described above,using conventional PCR cloning and ligation techniques.

Antibodies according to the present invention may be labelled with anyagent capable of generating a signal. The signal may be any detectablesignal, such as the induction of the expression of a detectable geneproduct. Examples of detectable gene products include bioluminescentpolypeptides, such as luciferase and GFP, polypeptides detectable byspecific assays, such as beta-galactosidase and CAT, and polypeptideswhich modulate the growth characteristics of the host cell, such asenzymes required for metabolism such as HIS3, or antibiotic resistancegenes such as G418.

The compositions containing the present selected antibodies of thepresent invention or a cocktail thereof can be administered forprophylactic and/or therapeutic treatments. In certain therapeuticapplications, an adequate amount to accomplish at least partialinhibition, suppression, modulation, killing, or some other measurableparameter, of a population of selected cells is defined as a“therapeutically-effective dose”. Amounts needed to achieve this dosagewill depend upon the severity of the disease and the general state ofthe patient's own immune system, but generally range from 0.00005 to 5.0mg of selected single chain antibody per kilogram of body weight, withdoses of 0.0005 to 2.0 mg/kg/dose being more commonly used. Forprophylactic applications, compositions containing the present selectedpolypeptides or cocktails thereof may also be administered in similar orslightly lower dosages.

A composition containing one or more selected antibodies according tothe present invention may be utilised in prophylactic and therapeuticsettings to aid in the alteration, inactivation, killing or removal of aselect target cell population in a mammal. In addition, the selectedrepertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the selected antibodies, cell-surface receptors orbinding proteins thereof whereby the undesired cells are killed orotherwise removed from the blood for return to the mammal in accordancewith standard techniques.

In a further aspect, the present invention provides the use of a singleheavy chain antibody as herein described as an intracellular bindingreagent.

Antibodies of the present invention can be expressed in any cell typeand may bind to and affect the function of any intracellular component.Intracellular components may be for example components of thecytoskeleton, molecules involved in gene expression and/or theregulation of expression, enzymes or molecules involved in theregulation of the function of cellular components. One skilled in theart will appreciate that this list is not intended to be exhaustive.Where for example the component is an enzyme inhibitor, an antibody ofthe present invention may increase or decrease the activity of theenzyme. The active site of enzymes is often located in the largestcavity on the protein surface. Such sites are not normally immunogenicfor conventional antibodies (Novotny et al, (1986) Proc. Nat. Acad USA,83, 226). The long H3 loop of single chain antibodies according to thepresent invention penetrates deeply into the active site of enzymes,allowing them to act as efficient enzyme inhibitors.

In particular the single chain antibodies, and/or fragments and/orcompositions thereof of the present invention may be of particular useas anti-viral and/or antibacterials in external applications, forinstance in the form of creams for skin, vaginal application and so on.In addition, antibodies fragments and compositions according to thepresent invention may find use in treating equipment, such as placeswhere opportunistic infections are prevalent. For example, antibodies,fragments thereof and compositions may be of particular use in hospitalenvironments, and in particular intensive care units. Furthermore, theantibodies, fragments thereof, and compositions of the present inventionmay find use in the treatment of transplantation material eitherartificial or natural tissue. For example stents or bone marrow infectedwith CMV or other viruses.

In addition, other functions may be added to antibodies of the presentinvention such as transport peptides and/or functional moietiesproviding an enzymic activity, for example kinases, proteases,phosphatases, de-acetylases, acetylases, ubiquitinylation enzymes,sumolation enzymes, methylases etc. Furthermore, other antibodies may beattached to the single chain antibodies, or fragments thereof accordingto the present invention. Those skilled in the art will appreciate thatthis list is not intended to be exhaustive.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry, molecular biology and biotechnology orrelated fields are intended to be within the scope of the followingclaims.

1-32. (canceled)
 33. A method for the production of a single heavy chainantibody comprising: (i) immunizing a transgenic rodent whose genomecomprises a heterologous VHH heavy chain locus with an antigen, whereinthe VHH heavy chain locus comprises: (a) at least one VHH exon, at leastone D exon and at least one J exon, wherein the VHH exon, the D exon andthe J exon are capable of recombining to form a VDJ coding sequence, andwherein the VHH exon encodes a camelid VHH, (b) at least one constantheavy chain region comprising at least one constant heavy chain gene,wherein each of said at least one constant heavy chain gene, whenexpressed, does not express a functional CH1 domain, and (c) a locuscontrol region providing for expression of the VHH heavy chain locusspecifically in B cells, wherein said mouse expresses the VHH heavychain locus in B cells in response to antigen challenge; and (ii)isolating single heavy chain antibody against said antigen.
 34. Themethod of claim 33, wherein the at least one D exon is of human origin,the at least one J exon is of human origin, and the constant heavy chainregion comprising at least one heavy chain gene is of human origin. 35.The method of claim 33, wherein the at least one constant heavy chainregion comprises at least one constant heavy chain gene which is not ofcamelid origin.
 36. The method of claim 35, wherein the at least oneconstant heavy chain gene which is not of camelid origin is of mouseorigin.
 37. The method of claim 33, wherein the entire VHH heavy chainlocus is of camelid origin.
 38. The method of claim 33, wherein theendogenous mouse heavy chain loci are deleted or silenced.
 39. Themethod of claim 33 wherein the rodent is a mouse.